Cells interact with structurally and biochemically distinct types of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat cell culture substrates? 2. What distinct signal transduction mechanisms control cell behavior in different 3D microenvironments? We are exploring whether classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We find differences in cell migration and signaling between 2D and 3D environments, but also between different 3D environments, such as collagen-based matrices that differ in architecture compared to fibronectin-rich cell-derived matrices. In these matrices, primary human dermal fibroblasts migrate at different speeds and with distinct modes of migration, with one major difference being the use of actin polymerization-based lamellipodial migration versus intracellular pressure-based lobopodial migration. For lobopodial migration, we had previously identified a contractility-dependent protein complex between the intermediate filament protein vimentin, actin, myosin II, and the nucleoskeleton-cytoskeleton linker protein nesprin 3. We collaborated with Michael Davidson to develop FRET-based biosensors to visualize these connections linking the extracellular matrix to the nucleus. Although the physical properties of extracellular matrices on flat (2D) extracellular matrices are known to modulate cell adhesion dynamics and overall cell migration, little is known about the roles of local micro-environmental differences between various types of 3D matrix on the dynamic behavior of living cells. We have generated 3D collagen gels of different matrix micro-architectures to search for differential regulation of 3D adhesion dynamics and cell migration. Collagen hydrogels polymerized at identical collagen concentrations but at different temperatures display dramatic differences in architecture, ranging from highly reticular with very short fibrils to large bundles of parallel collagen fibrils. Although classical measurements based on bulk rheology showed only minor differences in stiffness, more careful analyses of these matrices using atomic force microscopy probes at the micron-size scale of cell adhesions reveals that these fibrils organized in distinct architectures differ 10-fold in stiffness. Different collagen architectures frequently exist in close proximity in vivo, so these in vitro models can provide tractable systems for comparing cellular responses to each distinct type of architecture. Using atomic force microscopy, bundled collagen fibrils were found to be locally stiff at the size scale of cell adhesions, whereas reticular fibrils were soft. These 3D microenvironments were then used to compare effects of local stiffness on a variety of parameters of cell adhesion stability and rates of cell migration. Overall, cells migrating in 3D collagen appeared to require a local balancing of contractility with the specific local level of matrix stiffness through integrin receptors to stabilize cell adhesions in order to achieve the most efficient rate of cell migration. Interestingly, these human cells in the various 3D collagen environments displayed similar levels of integrin activation and clustering that were markedly elevated compared to the levels for the cells cultured on flat collagen matrices in cell culture. This enhanced integrin activation was accompanied by a requirement for cellular contractility, apparently in order for these cells to be able to detach effectively from their enhanced integrin-based attachments to 3D fibrils to mediate efficient cell migration. The next phase of this study will directly compare the interactions of malignant tumor cells with 3D fibrillar matrices, as well as the signaling required for efficient cell migration.